Extracellular vesicle proteomic analysis leads to the discovery of HDGF as a new factor in multiple myeloma biology

Abstract

Identifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.

Graphical abstract

Figure 1.

Mass spectrometry of proteins present in EVs released by IVPCs and HMCLs. (A) Graph showing the average number of peptides mapped to each protein upregulated in HMCL EVs relative to IVPCs. (B) Graph showing the average number of peptides mapped to each protein downregulated in HMCL EVs relative to IVPCs. Mass spectrometry analysis of EVs was done using independently harvested triplicates for each HMCL and IVPC generated from individual blood donors. Means ± standard deviation (SD) are shown. *P ≤ .01; others are P < .05.

Figure 2.

Plasma cell gene expression levels of differentially expressed proteins found in MM EVs. Analysis of archival gene expression profiles reveals that mRNA encoding 6 of the 9 HMCL EV target proteins are upregulated with increasing stages of malignancy. Significance was measured by one-way ANOVA.

Figure 3.

HDGF is present in HMCL extracts, HMCL EVs, and HMCL-conditioned media. (A) Top panel, Western blots demonstrate HMCLs express higher levels of HDGF than IVPCs; bottom panel, quantification of HDGF levels of expression normalized to total protein in each sample. Average of 3 experiments normalized to levels in KAS-6/1 (HMCL expressing the highest amount of HDGF). (B) Top panel, Western blots demonstrate EVs derived from HMCLs express varying amounts of HDGF, whereas EVs from IVPCs do not have detectable levels. Bottom panel, quantification of HDGF levels of expression normalized to total protein in each sample. Average of 3 experiments normalized to levels in DP-6 (HMCL EVs expressing the highest amount of HDGF). Nd, not detectable. (C) Levels of HDGF in HMCL-conditioned media measured by ELISA show that HMCLs secrete between 10- to 100-fold more HDGF than IVPCs. HMCL samples were tested in triplicate (except JMW and VP-6, which are duplicates); IVPC values reflect an average of 5 independent determinations. Means ± SD are shown.

Figure 4.

Autocrine effects of HDGF on HMCLs. (A) HDGF induces AKT phosphorylation in ALMC-1, ALMC-2, ANBL-6, and KAS-6/1 cells. Serum-starved cells were stimulated with IL-6 as a positive control and 50 ng/mL or 500 ng/mL HDGF for 1 hour. Top panels show the phosphorylation of AKT upon stimulation with HDGF; bottom panels show total AKT levels in each lane. siRNA knockdown of ALMC-1 (B,D) and ANBL-6 (C,E) results in reduced HDGF expression (B,C) and impaired proliferation (D,E). Results shown are representative of triplicate determinations. ***P < .001. Means ± SD are shown.

Figure 5.

HDGF enhances glycolysis and reduces mitochondrial respiration in ALMC-2 cells. (A) ECAR measurements show that both HDGF and IL-6 + HDGF increase ECAR. (B) HDGF increases ALMC-2 glycolysis. (C) OCR measurements show a significant reduction of mitochondrial respiration in the presence of HDGF. (D) HDGF significantly reduces the basal rate (BR), ATP production (ATP), maximal respiration (MR), and spare respiratory capacity (SRC). (E) HDGF reduces the OCR:ECAR ratio. (F) Quantitative analysis of FACS data showing that HDGF increases the number of cells with impaired mitochondrial membrane potential (TMRE). Oligomycin A (OmyA), 2-deoxy-d-glucose (2DG), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone/antimycin A (R/A), and tetramethylrhodamine, ethyl ester (TMRE). Results shown are representative of duplicate determinations (A-D,F) and triplicate determinations (E). Means ± standard error are shown (n = 3). *P = .05; ꬸP = .01; and ◊P < .001. A-D: t test comparing nil to all other conditions. F: t tests comparing HDGF to all other conditions.

Figure 6.

HDGF has a polarizing effect on macrophages. (A) HDGF induces an M1-like phenotype in human macrophages. Levels of CD80 (M1), CD206 (M2), and CD163 (TAM) expression following M1 polarization with LPS and IFNγ, M2 polarization with IL-4, or HDGF. Numbers represent the MFI ratio of each marker in each condition over media-only control ± SD. (B) Proteome profile of macrophages subjected to M1 and M2 polarizing cytokines as well as HDGF, showing that HDGF induces macrophages to secrete myeloma cell growth factor IL-6, as well as other proinflammatory cytokines and chemokines. Numbers for the triplicate experiments represent the ratio of each protein normalized to the corresponding media-only control.

Figure 7.

HDGF strongly reduces HLA-DR expression in human CD14⁺ monocytes and renders them immunosuppressive. Human CD14⁺ monocytes obtained from healthy volunteer blood donors were cultured with or without HDGF. (A) Representative histograms showing the reduction of HLA-DR when monocytes are cultured with HDGF (control monocytes are in red, HDGF-treated monocytes are purple). (B) Graph showing the percent inhibition of HLA-DR expression normalized to the levels of the control monocyte samples. (C) Representative T-cell proliferation histograms showing a significant reduction of T-cell proliferation when cocultured with HDGF-treated (HDGF-tr) monocytes. (D) Graph showing the percentage of dividing T cells in each condition. Means ± SD are shown (n = 4). *P < .001. Data shown in panels (B) and (D) represent the average values obtained from 4 independent experiments.